Abstract
The Institute of Evolution’s (IoE’s) Wild Lettuce Gene Bank (WLGB), established in the mid 1990s, contains new and extensive collections of five wild Lactuca relatives (WLRs) originating from Israel and Armenia: L. serriola, L. aculeata, L. georgica, L. altaica, and L. saligna. The objectives of the WLGB relate to the identification, collection, distribution, conservation, and characterization of the population genetic structure of these unique germplasms for crop improvement. Comprehensive studies are ongoing to determine the taxonomic position and crossing potential of the critical mass of collected species with domesticated lettuce, L. sativa, based on: (i) select morphological and phenological characteristics; (ii) molecular data; (iii) downy mildew resistance and (iv) variation in biologically active secondary metabolite content. In this review we present an overview of our key findings and highlight the advances in knowledge on these themes. Our germplasm collections and novel results, obtained by detailed, large-scale screening of natural populations and individuals for genetic variation, will considerably advance crop breeding research and practices. In addition, we critically summarize the recent literature and findings relating to three additional WLRs: L. dregeana, L. scarioloides, and L. azerbaijanica. The main long-term purpose of our research is to facilitate broadening of the genetic variation of domesticated lettuce by using new and adaptive germplasm in interspecific hybridization of lettuce.
Introduction
The genus Lactuca L. [Compositae (Asteraceae), tribe Cichorieae, subtribe Lactucinae] is comprised of 161 recognized species (WFO 2022), which are primarily found in the Northern Hemisphere (Lebeda et al. 2004a, 2014, 2019b, and literature cited therein). In the previous taxonomic classification of the genus Lactuca, species were organized into seven sections and two geographic groups, summarized by Lebeda et al. (2007a, 2009a). Section Lactuca L., sub-section Lactuca L. comprises the domesticated and cultivated species in the genus, Lactuca sativa L. (lettuce), together with ten wild species – Lactuca aculeata Boiss., Lactuca azerbaijanica Rech.f., Lactuca altaica Fisch. & C.A. Mey., Lactuca dregeana D.C., Lactuca georgica Grossh., Lactuca livida Boiss. & Reut., Lactuca saligna L., Lactuca scarioloides Boiss., Lactuca serriola L., and Lactuca virosa L. According to Prof. Daniel Zohary, who was a botanist with vast knowledge of the flora of Israel, the Near East, and the Mediterranean basin (Nevo 2017; Rottenberg 2017), a substantial part of these species is considered as closely related to the cultivated lettuce (Zohary 1991).
However, in the most recent comprehensive systematic classification of the Asteraceae family, the genus Lactuca was included in the subtribe Lactucinae of the tribe Cichorieae (Kilian et al. 2009). Lactucinae comprised approximately 200 species, with the greatest diversity located in southwest Asia and the Sino-Himalayan region (Kilian et al. 2009, 2017; Wang et al. 2013). In particular, Lactuca has been shown to be polyphyletic (Wei et al. 2016, 2017; Kilian et al. 2017). From the >100 species recognized within the genus Lactuca, ca 40. have been shown to represent a clearly monophyletic group, the “Lactuca lineage” (Kilian et al. 2017), including all examined North American species (Lebeda et al. 2019a, b).
The domesticated species in the genus, Lactuca sativa L. (lettuce), is one of the most important and widely distributed leafy vegetables across the world. Domestication has resulted in limited genetic variation, rending crop vulnerable to diseases, pests, and environmental stresses (Lebeda et al. 2014, 2022a). Therefore, breeders have stimulated the use of gene banks germplasm to promote food security and sustainable agricultural production (van Treuren et al. 2013).
Crop wild relatives (CWRs) are wild plant taxa genetically related to cultivated crops. They have potential use as gene donors in crop improvement programs due to their many desirable traits, such as resistance to pests and diseases or tolerance to abiotic stresses like drought, heat, and flooding (Taylor et al. 2017). Modern cultivars of most major crops already contain some genes from CWRs, and CWRs will continue to provide a viable and important source of genetic material to improve crop yields, enhance nutritional qualities, and modify husbandry requirements under future environmental changes (Ford-Lloyd et al. 2011). The linking of in-situ and ex-situ conservation with the use of CWRs, including wild Lactuca relatives (WLRs), is the leading principle of their conservation and management (Lebeda et al. 2009a, 2014, 2019a, b, and literature cited therein). Access to wild genetic resources and the possibility of exploring and exploiting them depend upon the successful protection of wild species in–situ, i.e., in their natural habitats, upon the complex study of wild species in natural habitats and upon information and biological material exchange prospects. Development of efficient conservation strategies for the maintenance of the highest possible genetic variability of crop progenitors is a key step in plant genetic resource management (Kitner et al. 2015). To this end, it is necessary to understand the genetic structure of progenitor species at both the species and population levels. Moreover, information about the distribution of genetic diversity among and within species/populations should serve as driving forces for collection and crop improvement strategies.
The continued improvement of lettuce by the creation of new varieties to ensure healthy, productive lettuce in the field and in the marketplace requires accessible, reliable sources of new genetic materials. These materials, called plant genetic resources (PGR) or germplasm, have diverse origins. Wild species closely related to domesticated lettuce (Wild Lactuca Relatives, WLRs) and primitive varieties of lettuce are found in many different countries (McGuire et al. 1993; Lebeda et al. 2007a, 2009a). The Institute of Evolution’s (IoE’s) Wild Lettuce Gene Bank (WLGB) in Haifa University (HU) was established by Prof. Eviatar Nevo, The IoE’s founder, in the mid 90’s. Twenty years ago, we initiated collecting and extensive studies on the population characterization structure of WLRs originating from Southwest Asia, that is the center of diversity for WLRs (Zohary 1991). Unique new collections of L. serriola, L. aculeata, L. georgica, and L. altaica [four of the seven wild Lactuca species, according to previous literature (Lebeda et al. 2007a, 2014; Zohary 1991), located in the primary lettuce gene pool (LGP1], and L. saligna (in LPG2) from Israel and Armenia were studied, as well as samples previously collected from Jordan, Turkey, and some other Mediterranean and European countries. We have also produced a minor collection of (1) four others wild Lactuca spp. (Lactuca viminea (L.) J. et C. Presl, Lactuca undulata Ledeb., Lactuca tuberosa Jacq., and Lactuca orientalis (Boiss.) Boiss.), that are from within the natural distribution region in Israel (Danin 2004; Feinbrun-Dothan 1978), but taxonomically far from cultivated lettuce and (2) two wild Cichorium (Compositae, tribe Cichorieae, a subtribe Cichoriinae) spp. (Cichorium pumilum Jacq., and Cichorium calvum Sch. Bip.). Figure 1 shows the number of accessions representing the collection of wild lettuces in IoE’s WLGB divided into four categories. The original seeds were collected from individual plants in their natural habitats, with each referred to as a sample (accession).
Number of accessions representing the collection of wild lettuces in IoE’s WLGB, divided into four categories: (i) core sub-subset: these were multiplied during ex situ seed regeneration and stored at -20oC, starting from 2015; (ii) core subset: the accessions in (i), plus accessions that were originally seed-collected and stored at -20oC, starting from 2015, plus accessions originally collected in Armenia in 2019 and stored at 4oC; (iii) core collection: accessions in (ii), plus accessions originally collected from 2007 and stored at 4oC; (iv) all collection: accessions in (iii), plus accessions originally collected from 1994, some of which were multiplied, and stored at 4oC.
Citation: Israel Journal of Plant Sciences 2023; 10.1163/22238980-bja10079
Number of accessions representing the collection of wild lettuces in IoE’s WLGB, divided into four categories: (i) core sub-subset: these were multiplied during ex situ seed regeneration and stored at -20oC, starting from 2015; (ii) core subset: the accessions in (i), plus accessions that were originally seed-collected and stored at -20oC, starting from 2015, plus accessions originally collected in Armenia in 2019 and stored at 4oC; (iii) core collection: accessions in (ii), plus accessions originally collected from 2007 and stored at 4oC; (iv) all collection: accessions in (iii), plus accessions originally collected from 1994, some of which were multiplied, and stored at 4oC.
Citation: Israel Journal of Plant Sciences 2023; 10.1163/22238980-bja10079
Number of accessions representing the collection of wild lettuces in IoE’s WLGB, divided into four categories: (i) core sub-subset: these were multiplied during ex situ seed regeneration and stored at -20oC, starting from 2015; (ii) core subset: the accessions in (i), plus accessions that were originally seed-collected and stored at -20oC, starting from 2015, plus accessions originally collected in Armenia in 2019 and stored at 4oC; (iii) core collection: accessions in (ii), plus accessions originally collected from 2007 and stored at 4oC; (iv) all collection: accessions in (iii), plus accessions originally collected from 1994, some of which were multiplied, and stored at 4oC.
Citation: Israel Journal of Plant Sciences 2023; 10.1163/22238980-bja10079
The IoE’s WLGB collections, accumulated over more than 20 years, have served as a basis for extensive theoretical research focusing on the identification, collection, characterization, conservation, and sustainability of these rich genetic sources for domestic lettuce improvement. These studies included: (i) eco-geographical distribution (Beharav et al. 2008, 2010a, 2018b); (ii) morphological and phenological diversity (Křístková et al. 2012; Lebeda et al. 2012a; Beharav et al. 2018a, b; Beharav and Hellier, 2020); (iii) genetic diversity (Sicard et al. 1999; Kuang et al. 2004, 2006, 2008; Kitner et al. 2008, 2015; Beharav et al. 2010b, 2018a; Lebeda et al. 2012a; Jemelková et al. 2015; Beharav 2022) (iv) lettuce downy mildew resistance (Beharav et al. 2006, 2014; Kuang et al. 2006; Petrželová et al. 2011; Lebeda et al. 2014; Jemelková et al. 2015; Beharav 2021) and virulence patterns of Bremia lactucae Regel, the causal of downy mildew (Sharaf et al. 2007); and (v) variations in composition and content of biologically active specialized natural products in several species of wild lettuces (Beharav et al. 2010c, 2015, 2020; Stojakowska et al. 2013, 2018; Michalska et al. 2014a) and Chicory (Michalska et al. 2014b). In order to maximize the above research, we initiated a collaboration with four research groups spearheading research in various topics relating to wild Lactuca spp.: (i) Department of Botany, Palacký University in Olomouc, Czech Republic; (ii) Department of Phytochemistry, Maj Institute of Pharmacology, Polish Academy of Sciences, Krakow, Poland; (iii) Plant Germplasm Introduction and Testing Research Unit, USDA-ARS,Washington State University, Pullman, WA; and (iv) The Genome Center and Department of Plant Sciences, University of California, Davis, CA, USA. Recently we initiated a new collaboration study with The College of Horticulture and Forestry Sciences, Huyazhong Agricultural University, Wuhan, People’s Republic of China. The main long-term purpose of our research is to facilitate broadening of the genetic variation of domesticated lettuce by using new and adaptive germplasm in interspecific hybridization of lettuce. Obtained results underscore the value of the above-mentioned species and WLRs for advancing research and for lettuce improvement (Lebeda et al. 2007a, 2014).
The aim of the present review is to critically summarize the available literature and our new insights from studies involving our WLRs collections and from the recent literature relating to three additional WLRs: L. dregeana, L. scarioloides, and L. azerbaijanica, thereby covering all species described by Zohary (1991) as the wild genetic resources of cultivated lettuce.
Characterization and evaluation of wild Lactuca species
L. serriola
Distribution
L. serriola (prickly lettuce) is known in two different forms, L. serriola f. serriola and L. serriola f. integrifolia with some specificity of their geographic distribution (Lebeda et al. 2001, 2004a, 2007b). Both forms are annual species widely distributed around the world, especially in the non-tropical parts of Eurasia, North Africa, and North and South America (Lebeda et al. 2004a, 2012b, 2019a, b, 2022b), growing mainly in disturbed areas such as waste places, field margins, roadsides, fallow and cultivated fields (Zohary 1991). In the Near East it occurs also in more primary habitats such as rocky gullies or open rocky slopes. L. serriola is considered to be the direct progenitor of cultivated lettuce (Zohary 1991; Lebeda et al. 2007a, 2022a) and its relationship to L. sativa is so close that they both are cross-compatible and inter-fertile (Zohary 1991, and literature cited therein).
Molecular studies
Genotyping over 1,000 accessions from IoE’s WLGB collection and originating all over the world using AFLP markers showed the differentiation of natural populations of L. serriola (Kuang et al. 2008). Wild Lactuca spp. showed rich diversity in disease resistance genes, as indicated by the high polymorphism of an SSR marker located in the coding region of the RGC2 resistance gene homologs (Sicard et al. 1999). Of the RGC2 family, some members were highly conserved among different accessions of L. serriola, while other members exhibited high divergence, with no obvious alleles in different accessions (Kuang et al. 2004). The characterized Dm3 gene, which belongs to the RGC2 family and encodes resistance against downy mildew (Bremia lactucae) (Parra et al. 2016), had 0.1% frequency in natural populations (Kuang et al. 2006). The Dm3 gene is infrequent in natural populations of L. serriola due to deletions and frequent gene conversions at the REC2 locus. By contrast, frequent chimera were detected in the 3’leucine rich repeat encoding region. Thus, the total number of resistant genes in a species may be very high. This has implications for the scales of germplasm conservation and exploitation of resistance sources (Kuang et al., 2006).
D’Andrea et al. (2017) also described high genetic differentiation among individual L. serriola populations, which can be ascribed to selection and colonization history (Nybom et al. 2014). A detailed analysis of AFLP polymorphism and isozyme variation of 50 European L. serriola populations, found their population clustering to correspond approximately to their geographical distribution (Lebeda et al. 2009b). The study also showed that accessions originating from various eco-geographical conditions differed significantly in their genetic and protein polymorphism, and in morphology.
The above-mentioned studies compared pseudopopulations representing individual plants collected from a large geographical area (Kitner et al. 2015). However, our detailed study conducted on L. serriola (and L. aculeata and L. saligna) in Israel, showed that, on a finer geographical scale, natural populations of wild Lactuca species are genetically well differentiated, and that each population can represent a unique combination of genotypes, which differ from other populations of the same species. Nevertheless, given the predominantly selfing character of L. serriola and other species, populations were not uniform (genetically or morphologically). It seems that, overall, genetic variation in a population increases at its distribution periphery, due to the presence of plants with ‘‘non-indigenous’’ alleles, which most likely arise from migration and subsequent interpopulation or interspecific hybridization (Kitner et al. 2015). These conclusions on population variation must be seriously considered when determining the spatial and temporal aspects of the L. serriola collection strategy. In addition, they emphasize the importance of collecting material from individual plants (e.g. Lebeda et al. 2001) and not as a mixture as was for long-considered an appropriate method (Guarino et al. 1995). These conclusions are directly supported by the resistance variation of L. serriola populations to Bremia lactucae (Petrželová and Lebeda 2011) and Golovinomyces bolayi (Lebeda et al. 2012c, 2013), and by the possibility of exploiting this variation in lettuce breeding (Lebeda et al. 2014; Parra et al. 2016).
Leveillula lactucae-serriolae on Lactuca serriola in Jordan
In our distribution survey of wild Lactuca in Jordan in August 2007, L. serriola plants with natural infections of powdery mildew, were observed at a site near Shobak (Ma’an Governorate) (Lebeda et al. 2019c). Characteristics of the asexual and sexual forms were obtained. Sequence analyses of the rDNA ITS region and D1/D2 domains of the 28S rDNA were used to obtain phylogenetic data, and to reach taxonomic conclusions about these specimens. Molecular determination, performed by sequencing of the ITS region, proved its identity with the type of material of Leveillula lactucae-serriolae (Lebeda et al. 2019c). This was the first taxonomically verified record of L. lactucae-serriolae on L. serriola growing wild in Jordan, and one of the first records of the fungus in the Near East (Voytyuk et al. 2009; Braun and Cook 2012; Lebeda et al. 2019c) and is an excellent example of the limited knowledge of plant pathogens in Near East on weedy growing Lactuca spp.
L. altaica
Proper placement in the LGPs according to previous literature
Based on crossing experiments using a sample named L. altaica (Lindqvist 1960a), Zohary (1991) concluded that L. altaica is a diploid 
wild lettuce. Fisher and Meyer (1846) described under the name L. altaica a form intermediate between L. saligna and L. serriola, originated from the Altai region. As Kirpicznikov (1964, 2000) notes, the morphological boundaries between L. altaica and these two species are sometimes blurred. Lindqvist (1960b) described various leaf feature that characterized some L. altaica sampled plants used in different studies. He concluded that there were some reasons for using the name L. altaica for a group of forms that were referred to as primitive forms of L. sativa. We believe the correctness of this approach. An alternative approach (e.g., Koopman et al. 1998) postulated that L. altaica be considered conspecific with L. serriola, which later brought the species to be described as a synonym of L. serriola in WFO (2022). To date, L. altaica has almost not been mentioned as a species studied by lettuce breeders and by crop evolutionists (Lebeda et al. 2007a, 2009b, 2014).
Morphological assessment
Seed samples of wild Lactuca spp. were collected by Dr. A. Beharav (IoE, HU, Israel), between August 30 and September 4, 2011, from 18 localities throughout four different regions of Armenia. Among the plants regenerated in a greenhouse from the original seed material that collected in Armenia in 2011, we have identified some which we believe are genuine L. altaica, according to their morphological characters. Stems of these plants were whitish and glabrous (Beharav et al. 2020), compared with yellowish (Feinbrun-Dothan 1978, p. 463) with medium density and semi-rigid trichomes that were observed in the lower half of stems of L. serriola plants (Beharav et al. 2018b). On the other hand, the flowering period of these plants was significantly shorter compared to that of L. saligna plants, while the number of ligules in the heads of these plants (averaged 22.9; ranged 16–34) were significantly higher compared to that of L. saligna plants (averaged 7.5; ranged 5–11, data not published). To confirm our taxonomic determination of these plants as L. altaica, morphological traits of many other identified L. altaica plants were carefully examined during a short expedition of A. Beharav, accompanied by Dr. Gayane Melyan, an expert from the scientific center of Agrobiotechnology, the Armenian National Agrarian University (ANAU), conducted on September 4–5, 2019, in 11 natural habitats, representing 5 different Armenian regions (Beharav et al. 2020).
Phytochemical studies
Initially phytochemical investigation of the roots of a single L. altaica sample, originally collected in Georgia in 1991, led to the isolation of 18 known sesquiterpenoids as well as 3-indolecarbaldehyde and seven known phenolic compounds (Michalska et al. 2010). The major sesquiterpene lactone found in the roots was lactuside A, which accounted for over 40% of the total amount of sesquiterpenoids. Notably, this compound also dominated the sesquiterpene profile in roots of L. sativa (Ishihara et al. 1987). Later, we performed a comparative phytochemical study of nine sesquiterpene lactones, based on 22 L. altaica plants, representing seven original individual seed samples derived from three localities representing three regions in Armenia (Beharav et al. 2020). The compounds were profiled and quantified in leaves and roots of the plants, grown in a controlled glasshouse. The contents of major sesquiterpene lactones, that including the following eight guaianolides: cichorioside B, lactucin, 11β,13-dihydrolactucin, crepidiaside B, 8-deoxylactucin, jacquinelin, lactucopicrin/11β,13-dihydrolactucopicrin, as well as the germacranolide glucoside – lactuside A, were estimated. The L. altaica plants could be characterized by the occurrence of lactuside A in their roots, and the mixture of lactucopicrin/11β,13-dihydrolactucopicrin in both their roots and leaves by relatively high amounts, similarly to results obtained for three commercial cultivars of L. sativa (Fig. 7). This study is likely the first report of detailed screening of L. altaica natural populations and individuals, even by low sample size, for any trait.
Proper placement in the LGPs according to our studies
To summarize, results obtained in the above studies strengthened our previous statement that, based on sesquiterpene lactone pattern, L. altaica shares more similarity with L. sativa than with L. serriola (Zidorn 2008; Shulha and Zidorn 2019) and with L. saligna. The view that the plant species that synthesize chemically complex pattern of metabolites are less advanced (Mabry and Bohlmann 1977) presupposes that L. altaica could be a primitive form of L. sativa. Notably, results obtained recently by various analyses [e.g., Neighbor-network cluster analysis (Fig. 5)] of the 115 KASP bi-allelic SNP genotyping assays (Beharav 2022) strengthened the other claims (e.g., Koopman et al. 1998) that L. altaica could be considered as conspecific with L. serriola. Clarification of the taxonomic status of L. altaica will require detailed studies of large sets of plant material originating from broader areas of the natural distribution of this species (e.g., Kirpicznikov 1964, 2000; Lebeda et al. 2004a).
L. aculeata
Distribution
L. aculeata is a robust and very prickly annual diploid plant 
, fully inter-fertile with L. sativa (Globerson et al. 1980) and with L. serriola, which can thus be included in the LGP1 (Zohary 1991; Lebeda et al. 2007a). Close relationships between L. aculeata and the crop complex were also supported by cases of spontaneous hybridization in their natural habitats (Lebeda et al. 2012a; Zohary 1991). According to Zohary (1991), L. aculeata distribution is restricted to the Near East and the Anatolian plateau. Israel is within the conjectured center of origin of L. aculeata, where this species occurs quite frequently, together with L. serriola and L. saligna, sometimes in the same habitats and mixed populations (Zohary 1991; Beharav et al. 2008, 2010a; Lebeda et al. 2012a; Kitner et al. 2015).
Eco-geographical characteristics and habitats
We recorded observations from a total of 60 native locations of L. aculeata throughout northern Israel (Beharav et al. 2010a, 2018b), which were mostly close to roadsides. This is likely the most comprehensive study undertaken to map populations of L. aculeata in a wider region of northern Israel, that enabled us to determine the boundaries of the L. aculeata habitats in Israel. The total of 60 locations represents the following five geographical districts of the Israeli flora: Mt. Hermon, Golan Heights, Hula Plain, Upper Galilee, and Lower Galilee (Fig. 2). Notably, our data included the first reported observations of native locations of L. aculeata on the western side of the watershed in Israel. The data indicate that the altitudinal range of L. aculeata in Israel is between 63 and 1268 m a.s.l.
Map of 60 observed locations of wild Lactuca aculeata throughout five geographical territories and districts of Israeli flora (according to Feinbrun-Dothan and Danin 1998) in northern Israel (Beharav et al. 2018b, Fig. 1).
Citation: Israel Journal of Plant Sciences 2023; 10.1163/22238980-bja10079
Map of 60 observed locations of wild Lactuca aculeata throughout five geographical territories and districts of Israeli flora (according to Feinbrun-Dothan and Danin 1998) in northern Israel (Beharav et al. 2018b, Fig. 1).
Citation: Israel Journal of Plant Sciences 2023; 10.1163/22238980-bja10079
Map of 60 observed locations of wild Lactuca aculeata throughout five geographical territories and districts of Israeli flora (according to Feinbrun-Dothan and Danin 1998) in northern Israel (Beharav et al. 2018b, Fig. 1).
Citation: Israel Journal of Plant Sciences 2023; 10.1163/22238980-bja10079
The area of five geographical districts in northern Israel could be considered as a hot spot for Lactuca spp. biodiversity in Israel. The main distribution of L. aculeta is basically in more xeric habitats, such as in Jordan and the Golan Heights, which are characterized by semi-humid to semi-arid climates approaching the Syrian and Jordanian deserts. In these xeric areas L. aculeata is very common. Generally, the trajectory from the Mediterranean eastwards towards the rift valley and Golan Heights is of increasing climatic aridity due to increasingly higher evapotranspiration, which results in increasing water deficits in the soil, even in high elevations in Mount Hermon where marginal Mediterranean and Iranoturanian vegetation may prevail. Cenomanian marls, and particularly Senoniam chalk, the latter weathering into rendzina drier soil, may explain the growth of steppic species in an otherwise Mediterranean climatic zone. For example, local microclimatic (Nevo 1995) and local edaphic divergence (Wang et al. 2018) may result in dramatic drought conditions that advance the colonization of steppes and adaptive drought-resistant plant populations as was shown in wild barley in the above cited papers.
Thus, the expansion of L. aculeata from the center of distribution in the east (Jordan, Mount Hermon and Golan Heights) to the west [the rift valley (Hula Valley), eastern Upper Galilee, and even Lower Galilee], may result from both regionally and locally coupled microclimatic and edaphic conditions. This regional movement was also recently reported coupled with expansion of the natural distribution area of L. aculeata to west of the Near East (Bergmeier and Meyer 2021), which can not be directly linked to changes in climatic or edaphic conditions, rather, to human migration (see above). However, clarification of taxonomic status of plants found by Bergmeier and Meyer (2021) on the islands of Limones and Lesbos (North Aegean region, Greece) will require more detailed morphological and molecular studies and assessment of their relationship to large collections of known L. aculeata and L. serriola. Notably, the plants classified as L. aculeata by Bergmeier and Meyer (2021), were described similarly to L. serriola plants with respect to two significant “quick field diagnostic” characteristics differentiating them from L. aculaeta plants (Beharav et al. 2018b): (i) the lateral branches of known L. aculeata plants are mostly horizontal to the main stem (Fig. 3a in Beharav et al. 2018b), in contrast to mostly acute angles of the lateral branches of the plants classified as L. aculeata by Bergmeier and Meyer (2021, Fig. 1), as well as of known L. serriola plants (Fig. 3b in Beharav et al. 2018b) and (ii) bright yellow florets of known L. aculeata plants (see Fig. 3a in Beharav et al. 2018b), in contrast to the pale-yellow florets of the plants classified as L. aculeata by Bergmeier and Meyer (2021, Fig. 1), and of known L. serriola plants (Fig. 3b in Beharav et al. 2018b). Indeed, features of trichomes on the stems of the plants classified as L. aculeata by Bergmeier and Meyer (2021, Fig. 1) typically seemed like those of known L. aculeata plants. At the same time, all plants we observed in a single location in Israel, that typically seemed L. serriola, contained trichomes characteristic of L. aculeata (Fig. 2 in Beharav et al. 2018b).
Out-crossing rate (t), estimated by average for whole polymorphic loci of wild Lactuca spp., using 11 EST-SSR loci (Kitner et al. 2015) and 115 KASP markers (Beharav 2022).
Citation: Israel Journal of Plant Sciences 2023; 10.1163/22238980-bja10079
Out-crossing rate (t), estimated by average for whole polymorphic loci of wild Lactuca spp., using 11 EST-SSR loci (Kitner et al. 2015) and 115 KASP markers (Beharav 2022).
Citation: Israel Journal of Plant Sciences 2023; 10.1163/22238980-bja10079
Out-crossing rate (t), estimated by average for whole polymorphic loci of wild Lactuca spp., using 11 EST-SSR loci (Kitner et al. 2015) and 115 KASP markers (Beharav 2022).
Citation: Israel Journal of Plant Sciences 2023; 10.1163/22238980-bja10079
Collecting, taxonomic validation and morphological assessment
Until recently, the germplasm of L. aculeata was very rarely found in genebank collections (Lebeda et al. 2004b) and has subsequently barely studied (e.g., Lebeda et al. 2002). This is the reason why our attention has focused not only on distribution and ecological studies (see above), but also on collecting achenes and creating a representative germplasm collection. The taxonomic status of a total of 185 L. aculeata sampled accessions was morphologically evaluated and validated during ex situ seed regeneration in 2008 (Beharav et al. 2010a) and 2015–2018 (Beharav et al. 2018b), together with 48 L. serriola sampled accessions (Beharav et al. 2018b), which followed standard seed-multiplication protocols for wild Lactuca species (Lebeda et al. 2007a). Propagation was carried out using single plant descent. Plants were grown in the greenhouse and were visually assessed at different developmental stages to focus on the best “quick field diagnostic” descriptions, differentiating between L. aculaeta and L. serriola plants, the two wild LGP1 spp., which naturally grow in Israel and Jordan. Accordingly, some hybrids of L. aculeata × L. serriola or their progenies were identified (Lebeda et al. 2012a). We observed variations in the phenology between the L. aculeata populations, but also between samples within populations, e.g., for plant height, days to flowering, and days to seed maturity. However, in general, flowering dates of L. aculeata plants were very late compared to that of L. serriola plants; In another greenhouse experiment (data not published) plants of L. aculeata accessions were significantly lately to bolt compared to plants of L. serriola accessions. Indeed, plants of L. aculeata accessions were also significantly lately to flowering compared to plants of L. serriola accessions originating from comparable habitats. Therefore, these traits could be mostly used as significant “quick field diagnostic” description between plants of the two species, highlighting those cases of rare overlapping may occur at the extremes. Recently, we initiated a study aiming to identify and clone genes that control the late flowering of L. aculeata plants.
Evidently, metapopulations of L. aculeata from Israel and Jordan seem to be more uniform in morphological characteristics than in their developmental characteristics. We have selected the following categorical characteristics as significant “quick field diagnostic” descriptions differentiating between L. aculeata and L. serriola plants (Beharav et al. 2018b):
- i.Leaf Features: In contrast to L. serriola, the whole leaf surface of L. aculeata plants is spinose-setose, as the species name implies. In general, L. aculeata plants mostly possessed an entire rosette and cauline leaves with obtuse apices. We rarely observed leaves dissected with dentate or serrate margins. In contrast, L. serriola plants only rarely possess rosette leaves. They generally possess divided cauline leaves, but sometimes also entire leaves; however, they absolutely differed from the spinose-setose leaves of L. aculeata plants.
- ii.Location of Trichomes on Stem Leaves: In all of the L. aculeata plants we observed, trichomes were located on the whole surface as well as on the main vein of the stem leaves, whereas in all of the L. serriola plants observed, the trichomes were located only on the main vein of the stem leaves.
- iii.Features of Trichomes on Stems: almost no variation was obtained for the observed L. aculeata plants. Dense (more than 10 trichomes/cm2) and rigid trichomes occurred along the whole stem of L. aculeata plants, while medium density (6–10 trichomes/cm2) and semi-rigid trichomes were observed only in the lower half of the stem of L. serriola plants.
- iv.Branching Angles and (v) Color of Florets: as described above.
- v.Color of Achenes: L. aculeata plants produced brown to bright brown achenes in contrast to greyish-brown achenes of L. serriola plants. Notably, according Güzel et al. (2021) L. aculeata can be distinguished by the glandular hairs on pedicels and the lax paniculiform synflorescence from L. serriola and L. sativa.
Molecular studies
As mentioned above, we used 11 EST–SSR loci and 230 AFLP markers to study the genetic structure of three wild Lactuca species (Kitner et al. 2015). Relatively low genetic diversity values were observed for L. aculeata, increasing in the following order: L. aculeata < L. serriola < L. saligna (Kitner et al. 2015). We have confirmed the assumption that L. aculeata as other WLRs are apparently a predominantly self-fertilizing plant (Fig. 3), using the EST-SSR loci (Kitner et al. 2015) and 115 KASP markers (Beharav 2022). However, our morphological (Fig. 4b, c) (Beharav et al. 2010a, 2018b; Křístková et al. 2012; Lebeda et al. 2012a) as well as isozyme studies (Lebeda et al. 2012a) showed evidence of the existence of spontaneous natural inter-specific hybrids between L. aculeata and L. serriola, where both species grow side-by-side in the same habitats (Fig. 4a). Nevertheless, cluster analyses of data obtained from our L. aculeata and L. serriola germplasm, based on some molecular markers, showed that individuals were subdivided into different clusters according to their taxonomic determinations (Lebeda et al. 2012a; Jemelková et al. 2015; Kitner et al. 2015; Beharav et al. 2018a; Beharav 2022)). However, the results of molecular analysis together with diagnostic distinguishing of morphological descriptions between the plants of the two species, suggest relatively very low levels of hybridization between the two-coexisting species in nature. This lies in agreement with data obtained from L. sativa and L. serriola under natural conditions, where hybridization rates were either low or highly dependent on distance between plants (D´Andrea et al. 2008).
(a) Natural field in Malkiya, Upper Galilee, Israel (33°06’10.4”N; 35°30’49.3”E; 668 m a.s.l.), showing coexistence of both L. aculeata (right) and L. serriola (left) plants; Results of natural hybridization between both parental species: (b) F1 plants (in bags); and (c) segregation of F2 family, progenies of individual L. aculeata plant no. 450–3 that were collected in Zefat, Upper Galilee, Israel (32°57’38.1”N; 35°30’53.7”E; 760 m a.s.l.) (Beharav et al. 2018b, Fig. 5)
Citation: Israel Journal of Plant Sciences 2023; 10.1163/22238980-bja10079
(a) Natural field in Malkiya, Upper Galilee, Israel (33°06’10.4”N; 35°30’49.3”E; 668 m a.s.l.), showing coexistence of both L. aculeata (right) and L. serriola (left) plants; Results of natural hybridization between both parental species: (b) F1 plants (in bags); and (c) segregation of F2 family, progenies of individual L. aculeata plant no. 450–3 that were collected in Zefat, Upper Galilee, Israel (32°57’38.1”N; 35°30’53.7”E; 760 m a.s.l.) (Beharav et al. 2018b, Fig. 5)
Citation: Israel Journal of Plant Sciences 2023; 10.1163/22238980-bja10079
(a) Natural field in Malkiya, Upper Galilee, Israel (33°06’10.4”N; 35°30’49.3”E; 668 m a.s.l.), showing coexistence of both L. aculeata (right) and L. serriola (left) plants; Results of natural hybridization between both parental species: (b) F1 plants (in bags); and (c) segregation of F2 family, progenies of individual L. aculeata plant no. 450–3 that were collected in Zefat, Upper Galilee, Israel (32°57’38.1”N; 35°30’53.7”E; 760 m a.s.l.) (Beharav et al. 2018b, Fig. 5)
Citation: Israel Journal of Plant Sciences 2023; 10.1163/22238980-bja10079
Downy mildew and powdery mildew(s) resistance
Because of the dire shortage of L. aculeata germplasms in genebanks (Lebeda et al. 2004b), it was impossible to perform more detailed resistance studies of this species (e.g., Lebeda et al. 2002, 2014; van Treuren at al. 2013). This was one of the considerations motivating our extensive L. aculeata collecting activities in Israel and surrounding (Turkey and Jordan) countries (Beharav et al. 2010a, 2018b; Lebeda et al. 2012a; Kitner et al. 2015). In natural habitats, L. aculeata is mostly free of any pathogen infection (Lebeda, unpublished data) and is not known as a natural host of B. lactucae (Lebeda et al. 2002). Because of easy crossability of this species with L. sativa, as a most interesting was considered resistance to Bremia lactucae. The existence of race-specific resistance to B. lactucae had been postulated for L. aculeata (Lebeda et al. 2002), and later preliminary studies (Beharav et al. 2006) confirmed this expectation. Later resistance studies were focused on the screening of larger number of accessions of L. aculeata for response to some Californian isolates of B. lactucae (Beharav et al. 2014); however, detailed information about race-specificity was still absent. In the most recent study, race-specific reaction patterns were frequently recorded, indicating the possible presence of some race-specific resistance factors/genes in the studied samples of L. aculeata (Jemelková et al. 2015). It is expected that this species could be a potential donor of resistance useful in lettuce breeding programs (Lebeda et al. 2012a, 2014; Jemelková et al. 2015; Beharav 2021).
However, we do not know anything about the mechanism of L. aculeata resistance to B. lactucae (Lebeda et al. 2008; Parra et al. 2016). Preliminary genetic studies have shown that one dominant R-factor could be expected in L. aculeata (Lebeda et al. 2002). Recent data led to the hypothesis that additional dominant race-specific resistance genes are localized in L. aculeata. This makes the broadly diversified germplasm of this species, growing in its center of origin around the Mediterranean Basin (Lebeda et al. 2004b), as an attractive and not well exploited resource for next research and lettuce breeding.
There is very limited knowledge on interactions between L. aculeata and powdery mildew. There are no available records of any powdery mildews on this species under natural conditions (Mieslerová et al. 2020), although there are several records of Golovinomyces bolayi on L. aculeata germplasm grown in the glasshouse in Europe (Mieslerová et al. 2020), i.e. outside of its natural distribution area. However, these preliminary observations showed that there could be rather large variation in natural resistance of this species to powdery mildew(s) (Lebeda, unpubl. data). The sources of resistance to this group of biotrophic pathogens remain to be identified in L. aculeata germplasm.
Variation of sesquiterpene lactones
A comparative phytochemical study of seven sesquiterpene lactones in natural populations of L. aculeata was performed, based on 23 accessions derived from eight, two, and single localities from Israel, Jordan, and Turkey, respectively (Beharav et al. 2010c). The compounds were profiled and quantified in leaves and roots of the plants, grown in a greenhouse under controlled conditions. L. aculeata was confirmed as a taxon strongly characterized by four dominant sesquiterpene lactones: 8-deoxylactucin, jacquinelin, crepidiaside B and lactuside A. An analysis of quantitative results of these four constituents led to the conclusion that obtained differences between and within populations are likely to be genetically controlled since all accessions were grown under standardized glasshouse conditions.
Future prospects
Variation obtained in the aforementioned studies, suggests that L. aculeata, a species belongs to the LGP1, harbours largely untapped genetic resources valuable for lettuce resistance breeding against biotic and abiotic stresses, and for promoting interesting agronomic traits that could potentially contribute to lettuce improvement. In particular, as a xeric species adapted to abiotic stresses, esp. high temperatures and low soil humidity, L. aculeata can serve as an important germplasm for drought resistance. The availability of our L. aculeata collections, which is likely the largest and most diverse collection, may be extremely valuable for both basic research and as novel genetic resources for breeding programs (Beharav et al. 2010a, c, 2014, 2018a; Lebeda et al. 2014; Jemelková et al. 2015).
L. georgica
Distribution
L. georgica, is a diploid (
) (Gabrielian and Zohary 2004; Zohary 1991), 50–300 cm tall, biennial plant according to Gabrielian and Fragman-Sapir (2008, p. 80). Blooming in nature occurs in July–August (Gabrielian and Fragman-Sapir 2008). Geographic distribution of L. georgica is restricted to the Euxinian-Hyrcanian region of Southwest Asia (Caucasia, Northeast Anatolia, and North Iran; Zohary 1991, and literature cited therein) and in the past, was observed at altitudes ranging from 1500 to 2300 m above sea level (Gabrielian and Fragman-Sapir 2008).
Molecular studies and proper placement in the LGPs
Seed samples of wild Lactuca spp. were collected by Dr. A. Beharav (IoE, HU, Israel), between August 30 and September 4, 2011, from 18 localities throughout four different regions of Armenia. We studied the genetic relationships of L. georgica samples originating in Armenia and the Russian Federation with samples representing four others predominantly self-pollinating wild Lactuca species (L serriola, L. aculeata, L. saligna, and L. virosa) originating in various countries, as well as with samples representing the cultivated lettuce, by using 48 Target Region Amplification Polymorphism (TRAP) markers (Beharav et al. 2018a). TRAPs are dominant markers that are generally distributed randomly across the genome of interest (van Treuren and van Hintum 2009). Our study appears to be the first more detailed molecular evaluation of L. georgica germplasm. Data analysis of the three major wild Lactuca species (L. georgica, L. virosa, and L. serriola) showed that allele frequencies of all 47 polymorphic loci varied significantly among the species. A total of 11, 9, and 10 alleles were unique to L. georgica, L. serriola, and L. virosa, respectively, 71% of TRAP marker diversity was between species. A Neighbor-Joining tree clearly clustered the whole set of 238 samples according to their taxonomic determination. The L. georgica samples clustered most distantly from the L. sativa. The interspecific comparisons between samples of L. georgica with L. sativa displayed a high genetic distance. A larger distance from L. sativa was only found in the comparisons with samples of L. virosa, that is species categorized into the LPG2 or LGP3 is not fully resolved yet (Lebeda et al. 2014).
Recently we studied the genetic relationships between 442 single-seed descent (SSD) lines, each representing a single accession from eight Lactuca spp., including five wild Lactuca relatives (WLRs) [Lactuca georgica, L. altaica, L. saligna, L. serriola, L. aculeata], L. tuberosa, L. undulata, and the domesticated lettuce, L. sativa, most of them (437) representing the core sub-subset of the IoE’s WLGB collection. The analysis was performed by profiling 115 single-nucleotide polymorphism (SNP) markers by means of the fluorescent KASP genotyping assay (Beharav 2022). The KASP marker fragments were scored as either allele “A” or allele “B”, that were used across analyses as bi-allelic data, but included relatively a lot of U-scores, i.e., an absence of the specific sequence, that were treated as missing data. Often U-scores were specific for a certain species. U-scores were obtained for 12.2%, 14.0%, 14.1%, and 15.1% of the L. sativa, L. serriola, L. aculeata, and L. altaica data points, respectively, while higher percentages (38.2% and 46.7%) of all the data points for L. saligna (LGP2) and L. georgica samples, respectively, resulted in U-scores. Highest percentages, 72.3% and 82.6% out of the whole data points resulted with U-scores for the L. tuberosa and L. undulata samples, respectively. Data analysis of the five WLRs showed that allele frequencies of 103 (97.2%) out of 106 differentiating loci varied significantly among the species, where 59.7% of the KASP marker diversity was between species. Two unique alleles were obtained for L. saligna, while a single unique allele was obtained for each of three species: L. georgica, L. serriola, and L. aculeata. The mean genetic distance was highest between interspecies pairs comprised of specimens belonging to L. georgica and samples belonging to the four LGP1 species. A neighbor-network analysis between samples belonging to the five WLRs and the single L. sativa cv. clearly clustered all 430 samples according to their taxonomic determination (Fig. 5). The results obtained here (Beharav 2022) via multiple complementary analyses of large natural populations and individuals for germplasm variation, question the assignment of L. georgica to the primary lettuce gene pool (LGP1). A similar conclusion was reached following a recently reported phytochemical study (van Treuren et al. 2018) and whole-genome resequencing (Wei et al. 2021). Together with our previous results obtained using TRAP markers (Beharav et al. 2018a), and hybridization experiments based on crosses between L. georgica and L. sativa that displayed only, but partially levels of interfertility (Beharav, personal communication), we conclude that L. georgica is a constituent of the LGP2, aligning with McGuire et al. (1993) concept of categorization of the Lactuca species into the various GPs, but in disagreement with Wei’s et al. (2021) recent statement, that L. georgica should be assigned to LGP3.
Neighbour-network cluster analysis based on uncorrected p-distance among 430 samples of six Lactuca spp., based on 115 KASP bi-allelic SNP genotyping assays (Beharav 2022, Fig. 1).
Citation: Israel Journal of Plant Sciences 2023; 10.1163/22238980-bja10079
Neighbour-network cluster analysis based on uncorrected p-distance among 430 samples of six Lactuca spp., based on 115 KASP bi-allelic SNP genotyping assays (Beharav 2022, Fig. 1).
Citation: Israel Journal of Plant Sciences 2023; 10.1163/22238980-bja10079
Neighbour-network cluster analysis based on uncorrected p-distance among 430 samples of six Lactuca spp., based on 115 KASP bi-allelic SNP genotyping assays (Beharav 2022, Fig. 1).
Citation: Israel Journal of Plant Sciences 2023; 10.1163/22238980-bja10079
Morphological assessment
Morphological observations supported the species identity of all L. georgica samples that we regenerated in the above-reported studies. The basic morphological features of the samples were distinctly different from samples representing the other four main WLRs collections in the IOE’s WLGB. In parallel, all L. georgica plants that we identified during the collection missions in natural habitats of Armenia, were clearly morphologically and taxonomically separated from all plants representing other WLRs. Thus, our observations are in contrast to the claim of Zohary (1991) that L. georgica plants show close morphological resemblance to L. serriola plants. Indeed, similar foliar, head and achene morphological features were displayed in our study by both L. georgica and L. virosa, but they differ by other minor morphological characters (Beharav et al. 2018a). The rosette leaves of L. georgica were not glaucous whereas the L. virosa samples were. For L. virosa the ligules colour was yellow to bright yellow, while the L. georgica plants had ligules colour of pale yellow to yellow. All L. virosa samples had smooth round stems, while all L. georgica samples also had smooth stems but with slight ribbing. A clear separation between these two species was obtained by the TRAP analysis (Beharav et al. 2018a) as well as previously by the sesquiterpene lactones composition (Michalska et al. 2014b). We have also distinguished between plants from some samples representing the two species by morphological features after five chlormequat (CCC; a growth retardant inhibiting GA biosynthesis; see Beharav et al, 1994, and literature cited therein) treatments of greenhouse grown plants (Beharav, unpublished data).
Bolting and flowering response to low temperatures
We demonstrated that low temperatures play a major role in stimulating the reproduction process of L. georgica plants (Beharav and Hellier 2020). Our results would suggest that for L. georgica: (i) there is an obligatory (or nearly so) vernalization requirement; (ii) plant age, vernalization duration, and genotype of original sample have a role in bolting and flowering regulation; (iii) some collected plants appear to behave as non-obligate typical biennials (but, most did), which is a commonly-accepted “fact” that has been cited for over one hundred years in the literature (Gabrielian and Fragman-Sapir, 2008, p. 80), but without any support of experimental data. Bernier et al. (1981) indicated that biennial plants with an obligate vernalization requirement normally undergo a juvenile phase during which they are insensitive to low temperatures. However, some of our tested germplasm, in which germinating seeds did respond to the vernalization treatment, do not fit to this scheme. (iv) four months of vernalization could be adequate to reach bolting in plants with a developed vegetative rosette, for most – but not all – samples; (v) in order to find the best solution for stimulating the reproductive process of multiple genotypes, it seems that further study should focus on about 4–6 months of vernalization at 4°C applied to plants of about 10–22 month old vegetative rosettes, with controlled post-vernalization condition; (vi) L. georgica germplasm could be used as a source for delayed bolting in breeding of lettuce varieties. High temperature induces early bolting and flowering in lettuce (Hong et al. 2013). So, increased temperatures from global climate change pose great challenges for lettuce production. Therefore, it is challenging to study the genetics of and the molecular mechanism underlying late bolting and flowering in WLRs to identify novel genes and alleles that were eliminated during lettuce domestication.
Resistance to B. lactucae
Seedlings of total 431 accessions representing Armenian natural populations of L. georgica and L. altaica, and mostly Israeli natural populations of L. saligna, L. serriola, and L. aculeata, were screened at seedling stage for resistance to six highly virulent races of B. lactucae (Beharav 2021). Our study is likely the first detailed screening of resistance to B. lactucae races of natural populations of L. georgica and L. altaica. The highest average of resistance probability (Fig. 6a) and frequency of highly resistant accessions (Fig. 6b) across races were detected in L. georgica. This is an interesting phenomenon as L. georgica is not known to be a natural host of B. lactucae (Lebeda et al. 2002) and from recent Bremia spp. taxonomical studies (Choi et al. 2011; Spring et al. 2018), it is evident that L. georgica can theoretically host different and unknown Bremia species.
Average resistance probability (a) and frequency of highly resistant accessions (b) of five wild Lactuca spp. to six Bremia lactucae races. Components of descriptive statistics for (a) are graphically presented: average value (horizontal short line), quartile range (rectangle), and data range (vertical long line) (Beharav 2021, Fig. 1).
Citation: Israel Journal of Plant Sciences 2023; 10.1163/22238980-bja10079
Average resistance probability (a) and frequency of highly resistant accessions (b) of five wild Lactuca spp. to six Bremia lactucae races. Components of descriptive statistics for (a) are graphically presented: average value (horizontal short line), quartile range (rectangle), and data range (vertical long line) (Beharav 2021, Fig. 1).
Citation: Israel Journal of Plant Sciences 2023; 10.1163/22238980-bja10079
Average resistance probability (a) and frequency of highly resistant accessions (b) of five wild Lactuca spp. to six Bremia lactucae races. Components of descriptive statistics for (a) are graphically presented: average value (horizontal short line), quartile range (rectangle), and data range (vertical long line) (Beharav 2021, Fig. 1).
Citation: Israel Journal of Plant Sciences 2023; 10.1163/22238980-bja10079
Frequency distribution for total sesquiterpene lactones content in roots (a) and leaves (b) of seven L. altaica sampled accessions from Armenia. Components of descriptive statistics are graphically presented in the upper part of the chart: Median value (vertical short line), 95% confidence interval (deltoid), quartile range (rectangle), data range (horizontal long line), and shortest half of the data (the densest region, red bracket). Perfomance of three cultivars of L. sativa are presenting by arrows (GL = Great Lakes; AB = Amerikanischer Brauner; GS = Grüner Stern) (Beharav et al. 2020, Fig. 3).
Citation: Israel Journal of Plant Sciences 2023; 10.1163/22238980-bja10079
Frequency distribution for total sesquiterpene lactones content in roots (a) and leaves (b) of seven L. altaica sampled accessions from Armenia. Components of descriptive statistics are graphically presented in the upper part of the chart: Median value (vertical short line), 95% confidence interval (deltoid), quartile range (rectangle), data range (horizontal long line), and shortest half of the data (the densest region, red bracket). Perfomance of three cultivars of L. sativa are presenting by arrows (GL = Great Lakes; AB = Amerikanischer Brauner; GS = Grüner Stern) (Beharav et al. 2020, Fig. 3).
Citation: Israel Journal of Plant Sciences 2023; 10.1163/22238980-bja10079
Frequency distribution for total sesquiterpene lactones content in roots (a) and leaves (b) of seven L. altaica sampled accessions from Armenia. Components of descriptive statistics are graphically presented in the upper part of the chart: Median value (vertical short line), 95% confidence interval (deltoid), quartile range (rectangle), data range (horizontal long line), and shortest half of the data (the densest region, red bracket). Perfomance of three cultivars of L. sativa are presenting by arrows (GL = Great Lakes; AB = Amerikanischer Brauner; GS = Grüner Stern) (Beharav et al. 2020, Fig. 3).
Citation: Israel Journal of Plant Sciences 2023; 10.1163/22238980-bja10079
Phytochemical studies
Our initial phytochemical investigation from roots of L. georgica plants, originally collected in September 2011 from a single locality in Armenia, led to the isolation of 15 sesquiterpene lactone aglycones and glycosides, including 10 lactucin-type guaianolides (Michalska et al. 2014a). This is the first report on the co-occurrence in Lactuca species of seven lactucin derivatives esterified at C-8 with acetic, methacrylic and 4-hydroxyphenylacetic acids. Therefore, the esters can be regarded as characteristic for L. georgica. The new natural product lactucin-8-O-methacrylate and its 11β,13-dihydroderivative were characterized in detail following a comparative analysis of their 1D- and 2D-NMR and high-resolution mass spectral data. Then, we performed a comparative phytochemical study of seven sesquiterpene lactones, based on 17 accessions derived from seven native locations representing three regions in Armenia (Beharav et al. 2015). The compounds were profiled and quantified in roots and leaves of the plants, grown in a glasshouse under controlled conditions. The contents of major sesquiterpene lactones were estimated in the plant materials by HPLC/PDA, including the germacranolide glucoside – lactuside A and the guaianolides: lactucin, 11β, 13-dihydrolactucin, its three esters (at C-8) with acetic, p-hydroxyphenylacetic and methacrylic acids and its 15-O-glucoside (cichorioside B). The plant roots could be characterized by the occurrence of lactuside A and two 11β, 13-dihydrolactucin derivatives (acetate and methacrylate) in relatively high amounts. Lactucin and 11β, 13-dihydrolactucin were major sesquiterpene constituents in the plant leaves. An analysis of quantitative results of these seven constituents led to the conclusions that obtained differences between and within populations are likely to be genetically controlled. This study is probably the first report of detailed screening of L. georgica natural populations and individuals for any trait.
Future prospects
Alongside their distinct floral habit, recent studies have suggested L. georgica as a new wild source of resistance to B. lactucae, their biochemical features, and their late bolting and flowering - highlight the uniqueness of this species. Even though recent results indicate that L. georgica probably belongs to the LGP2, we suggest that L. georgica should be considered as an attractive germplasm resource for lettuce breeding. Clearly, it justifies further identification and collection of additional L. georgica samples from multiple locations throughout its geographic distributions (Lebeda et al. 2004a; Zohary 1991). Following our experience, the optimal dates to collect ripe seeds of L. georgica in natural habitats is from mid-August to mid-September.
L. saligna
Eco-geographical distribution, collecting, taxonomic validation and morphological assessment
L. saligna (least lettuce, willow-leaf lettuce) is a weedy annual wild lettuce, primarily a Mediterranean species, occurring in Europe, northern Africa, and the Middle East (Lebeda et al. 2004a, 2007a; Beharav et al. 2008). However, it is also naturalized in North America (Lebeda et al. 2012b, 2019a,b), and also in Argentina and Uruguay (Monge et al. 2016), but not known from Chile (Lebeda et al. 2022b) in South America. It often colonizes roadsides, edges of cultivation, fallow, and waste places (Zohary 1991). Sometimes it grows side by side with L. serriola. Morphologically the differences between L. sativa/L. serriola and L. saligna are wider than those separating the crop complex from any other of the wild lettuces listed above (Zohary 1991). This wider taxonomic distance is also reflected cytogenetically. Fertile diploid hybrids between L. saligna and the crop were more difficult to obtain than after crosses between L. serriola and L. sativa (Lindqvist 1960a; Globerson et al. 1980), thus L. saligna can be included in the LGP2. Willow-leaf lettuce (L. saligna) is a modest-looking plant, with a large variation in fine details on leaves, stems and flowers, with rich internal features (e.g., secondary metabolites) as well as great variation in resistance to some biotrophic plant pathogens (Lebeda and Reinink 1994; Lebeda et al. 2002; Lebeda and Mieslerová 2011; Petrželová et al. 2011; Mieslerová et al. 2020), and thus an excellent source for lettuce breeding programmes for economically important features (Lebeda et al. 2014, 2016).
Seed samples from 562 individual plants that morphologically seemed to be L. saligna were collected from 41 localities, representing different climatic and edaphic environments throughout Israel. Intensive searching and collecting trips were conducted in September-October of 2004–2006 (Beharav et al. 2008), L. saligna plants were recorded throughout Israel except for desert areas (e.g., Negev and Judean desert) and extreme environmental/soil conditions (Dead Sea area). L. saligna was recorded at various altitudes (10 to 1277 m a.s.l.) and different habitats and soil types. In all, the taxonomic status of 214 of these accessions was morphologically validated as L. saligna during multiplication of 220 accessions in the greenhouse. In rosette formation and leaf morphology, different morphotypes of L. saligna were distinguished from the territory of Israel (Beharav et al. 2008), confirming previous reports relating to this species (Lebeda et al. 2016, and literature cited therein). Individual populations also varied in size, space structure and morphological uniformity. Our detailed population study showed that both of L. saligna populations exhibited higher diversity values, a greater number of SSR genotypes, including more plants with a heterozygous constitution, and higher number of private microsatellite and AFLP alleles, when compared to L. serriola and L. aculeata populations (Kitner et al. 2015).
Resistance to B. lactucae
Results obtained in our recent study (Beharav 2021) support previous findings (Lebeda 1986; Lebeda et al. 2002; Lebeda and Zinkernagel, 2003; Beharav et al. 2006, 2014; Petrželová et al. 2011) indicating that most accessions of L. saligna are highly resistant to B. lactucae, even at the seedling stage. However, the observations of some not fully resistant L. saligna - B. lactucae interactions at seedling stage may have been a plant stage-dependent effect. Quite a different situation was found at the adult-plant stage, where the observed resistance had a clear non-host character (Petrželová et al. 2011). Previous detailed histological and cytological studies of L. saligna – B. lactucae interactions showed that the formation of secondary vesicles represents the final stage of oomycete development, which is a characteristic of non-host resistance (Lebeda and Reinink, 1994; Lebeda and Pink, 1998; Lebeda et al. 2002, 2006). Also, biochemical (Sedlařova et al. 2007; Lebeda et al. 2008) and genetic (Jeuken and Lindhout, 2002) studies confirmed the status of this type of resistance which is very attractive in lettuce resistance breeding (Lebeda et al. 2014, 2016; Parra et al. 2016).
L. dregeana
According Zohary (1991) L. dregeana is a tall annual or biennial lettuce resembling L. serriola, but with smooth stem and lanceolate leaves. It is native to South Africa (Harvey and Sonder 1894), unlike the other wild Lactuca spp. described in the present review, which are of European or Asian origin. L. dregeana represents very probably a case of long-distance dispersal from Eurasia to the south tip of Africa (Zohary 1991). Since the morphological differences between this species and other members of the group described here are limited, this dispersal should have occurred in recent (geological) times. L. dregeana was for the first time recorded in east of Cape Town during 1772–1774 (Manning and van Herwijnen 2017). Investigation of the literature and herbarium collections suggests that L. serriola was also introduced to SA, but dates from the early twentieth century. The earliest record that has been found is from 1927 in the Fountains Valley in Pretoria, Gauteng (Manning and van Herwijnen 2017).
Plants originating from three accessions (09H5801127, 09H5801320, and PI 273574) supplied by the most renowned world Lactuca gene banks with labels of L. dregeana were grown and morphologically characterized during a field regeneration study conducted in 2009 at the IoE, HU, Israel, which followed all standard seed multiplication protocols (Lebeda et al. 2007a), except that seeds were not vernalized. All plants of the three L. dregeana accessions bolted, flowered, and had ripe seeds within several months (Beharav, unpubl. data), i.e., they appear to behave as obligate typical annuals.
While the name L. dregeana has been used or even extensively discussed in several recent studies (literature cited in Sochor et al. 2020), all (except for van Herwijnen and Manning 2017), including our observations mentioned above, were based on two or three gene bank accessions of unknown origin and dubious identity. This problem was identified by van Herwijnen and Manning (2017), who noted that the material in question was not an exact match of the wild L. dregeana collections. Lindqvist (1960b), who studied an accession from Modena Botanical Garden, Italy determined as L. dregeana, considered it to be a primitive form of L. sativa, based on some morphological characters (details in Sochor et al. 2020). After considering AFLP fingerprints and morphology, Koopman et al. (2001) also concluded that the material studied by them with respective labels of L. dregeana had likely escaped from cultivation.
In an effort to gain more insights on this taxon, recently Zeger van Herwijnen (Rijk Zwaan Breeding B.V., De Lier, The Netherlands), travelled extensively in SA to search the wild populations of L. dregeana and succeeded in locating it in several localities. According to van Herwijnen and Manning (2017) L. dregeana appears to be naturally uncommon and highly habitat specific species. They concluded that it represents a distinct genotype and recommend that it is accepted as a distinct species unless the study of wild-collected and properly authenticated material shows otherwise. To facilitate this, they summarised the available knowledge about the species and provided an updated description and distribution data (details in van Herwijnen and Manning 2017). Sochor et al. (2020) utilized for the first-time molecular methods, genome size estimation and phenotyping in the new wild-collected material of L. dregeana to shed light on the origin and evolutionary history of the species. Restriction-site associated DNA sequencing (RADseq), microsatellite (SSR) analysis and Sanger sequencing of the ITS region confirm that L. dregeana is a distinct taxon that has been derived from L. serriola approximately around the time of domestication of L. sativa. The new wild-collected samples of L. dregeana formed a separated cluster from control L. sativa accessions, while the gene bank accessions of primitive forms of L. sativa (formerly determined as ‘L. dregeana’) formed a distinct group close to other L. sativa accessions.
L. scarioloides
L. scarioloides is a second Irano-Turanian wild lettuce which shows close morphological affinities to L. serriola, from which it differs by the shape of the leaves, larger achenes and larger capitules. L. scarioloides is distributed over East Turkey, Iraqi Kurdistan, Iran and Afghanistan (Zohary 1991, and literature cited therein). It occurs mainly in elevated mountain ranges and on high plateaux (2000–3500 m altitude).
Recently, accessions of L. scarioloides collected in southeastern Turkey were subjected for the first time into a molecular phylogenetic study (Güzel et al. 2021). A total of 716 individual sequences belonging to 56 taxa of Southwestern Asian Lactucinae were generated. A single nuclear ribosomal and five plastid DNA markers were used for the tree’s construction. The three sampled L. scarioloides accessions were nested in the L. sativa clade (that include seven species: L. sativa, L. serriola, L. aculeata, L. saligna, L. virosa, L. georgica, and L. scarioloides) together with the L. georgica and L. virosa accessions in both, the nrDNA and plastid DNA gene trees, most closely to the L. virosa accessions (Güzel et al. 2021). Indeed, the mean interspecies genetic distance comparisons in this study of the whole set of the L. sativa clade samples, confirmed that L. scarioloides samples were most closer to the L. virosa, then to the L. georgica samples, as it seems in both trees. This question the placement of L. scarioloides in the LGP1 as it was described in previous literature (Zohary 1991; Lebeda et al. 2007a, 2014). Based on Thompson and Ryder (1961), that obtained successful hybridization between L. sativa and L. virosa only by chromosome doubling of the F1, McGuire et al.’s (1993) classified L. virosa into the LPG3. Recently Wood et al. (2020) reported on two lettuce lines that contain introgressed disease resistance from L. virosa, which was achieved using embryo rescue followed by extensive backcrossing to L. sativa (Maisonneuve 2003). However, Lebeda et al. (2014) noted that categorization of L. virosa into the LPG2 or LGP3 is not resolved yet. Thus, we assume the same status for L. scarioloides.
L. azerbaijanica
L. azerbaijanica is an endemic species date mentioned only from a single location in Iranian Azerbaijan (Rechinger 1977), so recently is accepted and considered as a native in NW Iran (Roskov et al. 2018). According Zohary (1991) morphologically it is closely related to L. scarioloides. So far, L. azerbaijanica has not been mentioned as a species studied by lettuce breeders and by crop evolutionists (Lebeda et al. 2014). To date, there are no detailed reports on collections of L. azerbaijanica samples in wild Lactuca gene banks throughout the world (Lebeda et al. 2004b, 2007a, 2009b). This species was not involved and considered also in recent taxonomical and phylogenetic studies of SW Asian Lactuca species (Güzel et al. 2021).
Summary
During the crop domestication process, only a part of the genetic diversity present in the gene pool of the wild species is included in the domesticated and cultivated crops, what is recently called as domestication syndrome (Smýkal et al. 2018). When looking for crop traits that add value to new varieties, nowadays breeders often turn to this reservoir of ‘left behind’ genes in the primary and secondary gene pools of their crop (Ondřej et al. 2021). Lettuce is one of the main horticultural crops where a strategy of wild related germplasm exploitation and utilization in breeding programs is most commonly used with very high practical impact (Lebeda et al. 2014). Our studies have identified rich genetic diversity between and within wild Lactuca species. Furthermore, we revealed that southwest Asia carries the most significant adaptive genetic diversity of WLRs germplasm, which can serve as donors of interesting traits into the domesticated lettuce. Developments in methodology of interspecific hybridization, as well as transfer of valuable alleles have been markedly facilitating access to the genetic variation present in wild Lactuca germplasm (Davey and Anthony 2011). For example, the late-flowering genes from L. aculeata, and the tolerance genes for biotic and abiotic stresses from some wild Lactuca spp. are valuable genetic resources for lettuce breeding programs. The rapidly developing genetics and genomics techniques will enable efficient identification of the target genes from wild species and their introgression into cultivated lettuce (Zhang et al, 2017; Wei et al. 2021), with parallel efforts to unlink unwanted genes (‘linkage drag’) from genes of interest (Ondřej et al. 2021). To conclude, the studies cited in the present review, based on the unique IOE WLGB collections, strengthened our previous statement that the collection, preservation, and utilization of the valuable WLRs described here are of cardinal importance for improving the domesticated lettuce varieties. Most important is the fact that many of the accessions cannot be obtained from their original source, i.e., they may be lost in their country of origin, discarded, or entirely inaccessible (McGuire et al.’s 1993). Therefore, most efforts soon should be devoted soon to increasing and balancing the number of accessions representing the core sub-subset [category (i) in Fig. 1] of the various wild lettuces in the IOE’s WLGB, using multiplication of single sampled accessions. Species with many accessions (see Fig. 1) can be partly multiplied as bulk samples from whole populations originally collected at individual location. This will complement our efforts for the benefit of global natural plant heritage and agricultural development.
Future prospects
Molecular and protein polymorphism
As WLRs represent an important source of genetic diversity, understanding their phylogenetic relationships with cultivated lettuce is of great practical importance in lettuce breeding. Molecular markers are powerful tools for estimating genetic diversity, since they are accurate, abundant, and unaffected by the environment. Several types of molecular markers have been analyzed in our and other studies of genetic relationships and structured diversity in natural population germplasms of wild Lactuca species surveyed in the present review. These studies carried out with AFLP (Kitner et al. 2008, 2015; Jemelková et al. 2015), allozyme loci (Dziechciarkova et al. 2004; Lebeda et al. 2012a), microsatellites (Jemelková et al. 2015; Kitner et al. 2015; Sochor et al. 2020), TRAP (Beharav et al. 2018a), nuclear ribosomal and/ or plastid DNA markers (Sochor et al. 2020; Güzel et al. 2021), RADseq (Sochor et al. 2020), and KASP markers (Beharav 2022). Notably, we concluded that KASPs assays are not the best marker system for analysis of wild Lactuca spp. representing different LGPs, when markers were chosen from samples representing species belonging to LGP1 (Beharav 2022). Clearly, this conclusion can serve as a general conclusion for studies evaluating the genetic diversity of species from other taxa.
The phylogenetic trees obtained in the above studies generally clearly clustered the entire set of samples according to their taxonomic determination. To summarize, of the seven wild species previously presumed to constitute the LGP1 (Zohary 1991; Lebeda et al. 2007a), four (L serriola, L. altaica, L. aculeata, and L. dregeana) indeed belong to LGP1, while L. georgica belongs to LGP2, and L. scarioloides to LPG2 or LGP3. We assume the same status (LPG2 or LGP3) for L. azerbaijanica, since it is morphologically closely related to L. scarioloides (Zohary 1991). L. saligna was assigned to LGP2, aligning with earlier reports. The above conclusions are based on the combination of: (i) molecular phylogenetic trees that were described in the present review, (ii) genetic distance matrixes of interspecies comparisons in the molecular studies, and (iii) the concept of categorization of the Lactuca species into the various GPs, based on the level of success of their crosses with domesticated lettuce and fertile F1 seed yield, as described by McGuire et al.’s (1993, and literature cited therein).
Morphological and phenological variation
In addition to the DNA-based marker data, morphological traits are of great help in evaluation genetic resources (Šuštar-Vozlič et al. 2021; Lebeda et al. 2022a). Although morphological characters not always provide clear answers, due to ambiguous differences or phenotypic modifications caused by environmental factors (Garcia et al. 2002), we are exploring traits that are known to be very stable under various environmental conditions and could therefore be employed as an identification tool as well as source of information concerning adaptive genetic diversity. Such morphological information gathering at both the intra, and inter-species levels can help refine seed collecting strategies, and widen the diversity maintained in germplasm collections for crop improving.
A large degree of variation in plant phenotypes has been observed and described in a greenhouse experiment among 68 samples of the three WLRs distributing in Israel, L. serriola, L. saligna, and L. aculeata (Lebeda et al., data not published). Seeds for this study were collected from individual plants in northern Israel, along a line transect, and two populations per each Lactuca species. Assessments included 28 characters of stem, rosette and cauline leaves, inflorescence, flowering, and flowers, as following: 25 categorical (qualitative) variables: 21 out of them are nominal (rosette development; position of rosette leaves; rosette leaf – lamina, depth of incisions, shape of apex, and anthocyanin location; entire rosette leaf - shape in outline; stem leaf – lamina, shape of apex, anthocyanin location, and trichomes location; entire stem leaf - sheape in outline; divided stem leaf – depth of incicions; stem – way of branching, localization of trichomes, and quality of trichomes; type of composed inflorescence; flower – colour of ligule, anthocyanine in anther tube, and location of anthiocyanin on ligule; and involucral bracts – location of anthoicyanin) while four are ordinal variables (stem leaf – intensity of anthocyanin coloration; branching – proportion between the branched part and the whole stem; stem – density of trichomes; and flower – intensity of anthocyanin coloration on ligule), as well as three continuous (quantitative) variables: number of flowers per capitula; days to bolting; and days to flowering). The species identity of all studied accessions was taxonomically verified according to the 28 characterized traits. The features of three nominal characters were successfully measured in plants of only a single species. For two other nominal characters, only a single category level was observed for the entire sample set. For 19 out of the remaining 20 categorical characters, significant differences were noted across the three WLRs. Significant differences between species were also noted for the three evaluated quantitative characters.
Downy mildew resistance
Breeders have used wild lettuce spp. germplasm as sources of disease resistance (Lebeda et al. 2007a). Most efforts have been devoted to B. lactucae (Lebeda et al. 2002), the causal of lettuce downy mildew, which is the most harmful disease of lettuce worldwide. The study of its biology and epidemiology, source of resistance, mechanisms and genetic control of resistance in WLRs have been a high priority of researchers and breeders in many countries (Lebeda et al. 2014, and literature cited therein). Most of the resistances against B. lactucae are race-specific and determined by dominant resistance genes (Dm or R-factors) (Michelmore et al. 2009). Precise knowledge of the location of new Dm genes provides the foundation for marker-assisted selection to breed cultivars with multiple genes for resistance to downy mildew (Parra et al. 2016, 2021).
Seedlings of total 431 accessions representing five WLRs were recently screened at seedling stage for resistance to six highly virulent races of B. lactucae (Beharav 2021). To facilitate quantitative analysis of accession resistance scores +, (+), +/-, (−), and –, were transformed to numerical values of resistance probability, i.e., 0.0 (highly susceptible), 0.25, 0.5, 0.75 and 1.0 (highly resistant), respectively. The frequencies of the five resistance scores highly differed across Lactuca species. L. aculeata showed the highest resistance probabilities among the three wild species belonging to the LGP1 (L. aculeata, L. altaica, and L. serriola) examined in this study. This study supports our previous studies (Beharav et al. 2014; Jemelková et al. 2015) which showed complete resistance of some L. aculeata samples to a set of highly virulent B. lactucae isolates. For L. serriola, the closest relative of cultivated lettuce, and for L. altaica, also a closer or even a primitive form of L. sativa, probabilities of resistance to each of the investigated B. lactucae races were very low (Beharav 2021).
Obtained recent results (Beharav 2021) support our previous findings (Lebeda 1986; Lebeda et al. 2002; Beharav et al. 2006, 2014; Petrželová et al. 2011) indicating that most accessions of the known non-host resistance (NHR) species L. saligna, which classified to the LGP2, are highly resistant to B. lactucae, even at the seedling stage. However, the highest average resistance probability and frequency of highly resistant accessions across the six B. lactucae races tested in the recent study (Beharav 2021) were detected in L. georgica. On accession level, some moderate resistance (scored 0.5), and even a single event of less than moderate resistance (scored 0.25), was observed for some race-L. saligna accession interactions. For L. georgica, most of the tested accessions expressed highly resistant probability (scored 1.0) for each of the six races, while only some sparse sporulation (resistant probability scored 0.75) was observed in some race-L. georgica-accession interactions. Thus, we suggest that L. georgica is a new LGP2 source of resistance to B. lactucae. However, there is a general question about the definition of that L. georgica resistance: is it a NHR or a highly resistance in most accessions of L georgica? In the definition of NHR plant species, “all genotypes of a plant species are resistant to all genotypes of a pathogen species” (Heath, 1991). Therefore, the sporulation level in adult L. georgica plants should be evaluated. Some partially resistant (scored 0.75) L. georgica–B. lactucae interactions obtained in our recent study at seedling stage (Beharav 2021), may be plant stage dependent, as was confirmed for L. saligna (Zhang et al. 2009; Petrželová et al. 2011; Beharav et al. 2014; Giesbers et al. 2018). Notably, at the adult-plant stage, none of L. saligna accessions were susceptible to any of the isolates tested in our previous studies (Petrželová et al. 2011; Beharav et al. 2014). Thus, we hypothesize that L. georgica, which classified to the LGP2, will also classify as a NHR species after adult plants from the most comprehensive set of sampled accessions will evaluate to some B. lactucae isolates.
To summarise, while resistance among L. georgica and L. saligna (LGP2) to highly virulent races was prevalent, also some L. aculeata (LGP1) accessions showed broad resistance to a set of highly virulent B. lactucae isolates, suggest that the germplasm of these three WLRs may serve as a novel model for assessing the transient expression of candidate B. lactucae effector genes (e.g., Giesbers et al. 2017), for identifying, fine mapping, and cloning loci and genes conferring resistance to B. lactucae, and later applying them for breeding resistance into domesticated lettuce.
Recently, the structure, complexity, differences and connections between wild plant pathosystems (WPPs) and crop plant pathosystems (CPPs) were demonstrated through a case study of lettuce (Lactuca spp., L. serriola and L. sativa) and lettuce downy mildew (Bremia lactucae) (Lebeda and Burdon 2023). Research on interactions between plant pathosystems, the “cross-talk” of WPPs and CPPs, is still very limited. However, this knowledge could be seriously considered and applied in plant breeding and disease management (Lebeda and Burdon 2023).
Variation in biologically active secondary metabolite content
In recent years, a number of lettuce cultivars have been studied chemically and found to contain biologically active specialized natural products, including phenolic compounds and sesquiterpenoids, can be beneficial for human health (Chadwick et al. 2013; Yang et al. 2022). Some of the compounds are of common occurrence in the genus Lactuca (e.g., caffeoylquinic and caffeoyltartaric acids, quercetin and luteolin derivatives) while the others can be found only in some species (coumarins, lignans, and sesquiterpene lactones of different structures). Chemical composition of wild Lactuca species is expected to be a substantial factor in disease resistance (Bennett et al. 1994), useful for germplasm evaluation and lettuce breeding. Different taxa from some sections of the genus Lactuca have been reported to produce a variety of triterpen and sesquiterpene lactones, including guaianolides, eudesmanolides and germacranolides as well as antioxidant phenolic compounds (Beharav et al. 2020, and literature cited therein). The sesquiterpene lactone lactucin and its derivatives are well known constituents of Cichorium and some Lactuca species (Zidorn 2008). In particular, lactucin-type guaianolides (e.g., 8-deoxylactucin, lactucin, lactucopicrin together with their 11,13-dihydroderivatives) have been recognized as characteristic specialized natural products of the plants that are used as popular vegetables. Except for the lactucin-type guaianolides that are constitutively present plant metabolites, lettuce plants synthesize guaianolide phytoalexins, lettucenins, in a response to fungal or bacterial infection (Bennett et al. 1994, 2022; Sessa et al. 2000). The phytoalexins seem to share their biosynthetic pathway with the constitutively present compounds. Detailed molecular mechanisms of the biosynthetic pathway regulation and relationships between the pools of constitutively present and induced guaianolides in lettuce plants have not been worked out as yet (Mai and Glomb 2016). Lactucin-type sesquiterpene lactones are for the most part responsible for the characteristic bitter taste of the lettuce and chicory plants (van Beek et al. 1990; Mai and Glomb 2016). Their metabolic function has been strongly implicated in the plant defence response (Beharav et al. 2010c, 2015, and literature cited therein), especially against insect pests, but seems to play marginal role in virus and fungal resistance of lettuce plants. Thus, it appears that sesquiterpene lactones represent a good target for future genetic manipulation of cultivated lettuce leading to an enhanced protection of the plant and disease resistance. Such genetic material could serve as an excellent genetic model system for studying the genetic factors controlling biochemical pathways. Due to frequent use of interspecific hybridization in lettuce resistance breeding programs, some elevation of sesquiterpene levels in cultivated lettuces should be considered. However, those levels should not exceed acceptable level from the viewpoint of tasting and eating quality, which is the priority.
The integration of chemophenetic data to the botanical systematics is limited by the lack of standard specimens for verifying the identity of plant material (Zidorn 2008). When correctly identified plant material is available, the results of chemical analyses bring a new light to evolutionary concepts and taxonomical relationships between different Lactuca spp. (Lebeda et al. 2014, and literature cited therein). Results obtained in our phytochemical studies strengthened previous taxonomic concepts of the sampled accessions that were based on morphological traits and molecular diversity of the tested species (Beharav et al. 2010c, 2015, 2020). Phenolic compounds and triterpenoids synthesized by WLRs, due to their common occurrence in Lactuca spp. and some other taxa are not useful as chemical markers within the genus. Sesquiterpene lactones seem to be the best suited for the purpose. The first attempt to associate the sesquiterpene lactone pattern with taxonomic classification within the Lactuca genus was made in 2009 (Michalska et al. 2009). Six members of the section Lactuca L., subsection Lactuca L. (L. aculeata, L. sativa cv. British Hilde, L. serriola f. serriola, L. saligna, L. virosa var. virosa, ‘L. dregeana’), two species from the section Lactuca L. subsection Cyanicae DC. (Lactuca perennis L., Lactuca tenerrima Pourr.), and three species representing sections: Mulgedium (Cass.) C.B. Clarke (Lactuca tatarica (L.) C.A. Meyer), Tuberosae Boiss. (Lactuca indica L.), and African group (Lactuca inermis Forssk.) were included into the study. In the light of the present knowledge, the examined ‘L. dregeana’ plants were most probably not the representatives of the rare endemic species but a primitive form of L. sativa (Sochor et al. 2020), which is in agreement with chemical data. Further research on sesquiterpene lactones from Lactuca spp. (L. aculeata, Lactuca tenerrima Pourr., L. inermis, L. altaica, L. georgica, L. viminea, Lactuca canadensis L., L. orientalis, L. tuberosa, Lactuca plumieri (L.) Gren. & Godr., L. undulata), conducted following a uniform methodology, confirmed that the taxa included into subsection Lactuca L. can be characterized by the presence of easily detectable lactucin and 8-deoxylactucin derivatives in both aerial parts and roots of the plants and by the occurrence of the germacranolide lactuside A that is accumulated exclusively in roots of the plants (Stojakowska et al. 2018; Shulha and Zidorn 2019; Michalska et al. 2021). Outside the subsection Lactuca, the latter compound was found only in a few species including L. viminea (section Phaenixopus (Cass.) Benth.), L. indica (section Tuberosae), and L. perennis (subsection Cyanicae). It is worth noting that some species of wild lettuces i.e.: L. tenerrima (subsection Cyanicae; Michalska et al. 2012), L. undulata (section Micranthae Boiss.; unpublished results) and L. tuberosa (Stojakowska et al. 2013) seem to not produce sesquiterpene lactones at all.
Pharmacological exploitation of some chemical constituents of cultivated and wild lettuce plants is another goal of studies aimed at their detection and characterization (Lebeda et al. 2014, and literature cited therein). Potential benefits to human health due to lettuce consumption have been recently critically reviewed by Yang et al. (2022). A lot of research is focused on commonly found polyphenols and their antioxidant activity. Health benefits brought about by the lettuce terpenoids are much less studied. Carotenoids, except for their antioxidant activity (Bohn 2019) are important source of provitamin A. Their degradation products, apocarotenoids, isolated from stem lettuce, L. serriola and L. indica, are also biologically active as allelochemicals and anti-inflammatory, chemopreventive and antihypertensive agents (Kim et al. 2008; Malarz et al. 2021 and literature cited therein). Not much is known about triterpenoids synthesized by WLRs and their activity. Pentacyclic triterpenes are known active constituents of cultivated lettuces (Choi et al. 2022). Very recently, inhibition of colorectal cancer cell proliferation by taraxasterol acetate has been described by Tang et al. (2021). Lactucin-like guaianolides, regarded as active components of lactucarium (dry milky sap from some wild lettuces; Bynum and Bynum 2016), exert antinociceptive and sedative effect (Wesołowska et al. 2006, and literature cited therein). Lactucopicrin is currently the most frequently investigated compound of this type. The lactone effectively ameliorated oxidative stress in scopolamine-treated rat glioma C6 cells, reversed toxic effects induced by scopolamine treatment in murine neuroblastoma N2a cells and stimulated neuritogenesis (Venkatesan et al. 2016, 2017). Dietary supplementation with lactucopicrin may lead to the inhibition of vascular endothelial NF-κB activation and consequently to mitigation of the vascular inflammatory diseases (Weng et al. 2021). Thus, pure chemical components and chemically defined active fractions of extracts from lettuce plants may find use as dietary supplements to prevent lifestyle diseases widespread in post-industrial society.
Acknowledgements
Studies described in this review were financial supported by grants from the: (i) Research Authority, University of Haifa, Israel; (ii) Israel Ministry of Science; (iii) Internal Grant Agency of Palacký University in Olomouc, Czech Republic (IGA-PRF-2022-002; IGA-PRF-2023-001); (iv) Czech Ministry of Education, Youth and Sports; (v) Czech Ministry of Agriculture; (vi) Poland Ministry of Science and Higher Education; (vii) USDA-ARS CRIS Project and NIFA MultiState Project; (viii) California Leafy Greens Research Board; and (ix) Ancell-Teicher Foundation for Molecular Genetics and Evolution. AB wishes to thank: (i) Some Armenian colleagues (mentioned in Beharav et al. 2015, 2018a, 2020) for their excellent organization, common identification and collection of unique wild Lettuce’s germplasm in 2011 and 2019; (ii) Rijk Zwaan Breeding B.V., De Lier, The Netherlands, for providing greenhouse facilities, support in assessment and cultivation of approximately 500 sampled Lactuca accessions (see Beharav 2021, 2022; Beharav et al. 2018b, 2020), screening for resistance to six B. lactucae races (Beharav 2021), as well as providing and explanation of KASP data points (Beharav 2022); (iii) Dr. Murat Güzel, Department of Biology, Faculty of Science, Karadeniz Technical University, Trabzon, Turkey, for providing the genetic distance matrixes of his study (Güzel et al 2021); (iv) Prof. Hanhui Kuang, College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan, People’s Republic of China, for contribution of an important paragraph that summarized results of some preliminary studies using germplasm from the IoE WLGB collections, and for initiating a new collaboration on the late-flowering genes from L. aculeata; and (v) Ms. Souad Khalifa from the IOE for her excellent technical contribution along the years. The authors declare no conflict of interest regarding the publication of this paper.
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